Ebola peptides and immunogenic compositions containing same

ABSTRACT

Using CTL epitopes to the  Ebola  GP, NP, VP24, VP30, VP35 and VP40 virion proteins, a method and composition for use in inducing an immune response which is protective against infection with  Ebola  virus is described.

BACKGROUND OF THE INVENTION

This application is a continuation-in-part of U.S. utility applicationSer. No. 09/337,946, filed Jun. 22, 1999 now abandoned, which claimspriority from U.S. provisional application 60/091,403 (filed Jun. 29,1998). The entire contents of both applications are incorporated hereinby reference.

Ebola viruses, members of the family Filoviridae, are associated withoutbreaks of highly lethal hemorrhagic fever in humans and nonhumanprimates. The natural reservoir of the virus is unknown and therecurrently are no available vaccines or effective therapeutic treatmentsfor filovirus infections. The genome of Ebola virus consists of a singlestrand of negative sense RNA that is approximately 19 kb in length. ThisRNA contains seven sequentially arranged genes that produce 8 mRNAs uponinfection (FIG. 1). Ebola virions, like virions of other filoviruses,contain seven proteins: a surface glycoprotein (GP), a nucleoprotein(NP), four virion structural proteins (VP40, VP35, VP30, and VP24), andan RNA-dependent RNA polymerase (L) (Feldmann et al.(1992) Virus Res.24, 1-19; Sanchez et al.,(1993) Virus Res. 29, 215-240; reviewed inPeters et al. (1996) In Fields Virology, Third ed. pp. 1161-1176.Fields, B. N., Knipe, D. M., Howley, P. M., et al. eds. Lippincott-RavenPublishers, Philadelphia). The glycoprotein of Ebola virus is unusual inthat it is encoded in two open reading frames. Transcriptional editingis needed to express the transmembrane form that is incorporated intothe virion (Sanchez et al. (1996) Proc. Natl. Acad. Sci. USA 93,3602-3607; Volchkov et al, (1995) Virology 214, 421-430). The uneditedform produces a nonstructural secreted glycoprotein (sGP) that issynthesized in large amounts early during the course of infection.Little is known about the biological functions of these proteins and itis not known which antigens significantly contribute to protection andshould therefore be used to induce an immune response.

Recent studies using rodent models to evaluate subunit vaccines forEbola virus infection using recombinant vaccinia virus encoding Ebolavirus GP (Gilligan et al., (1997) In Vaccines 97, pp. 87-92. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.), or naked DNAconstructs expressing either GP or sGP (Xu et al. (1998) Nature Med. 4,37-42) have demonstrated the protective efficacy of Ebola virus GP inguinea pigs. (All documents cited herein supra and infra are herebyincorporated in their entirety by reference thereto.) Additionally,Ebola virus NP and GP genes expressed from naked DNA vaccines(Vanderzanden et al.,(1998) Virology 246, 134-144) have elicitedprotective immunity in BALB/c mice. There has been one study that showedprotection in nonhuman primates with a high dose DNA prime/high doseadenovirus boost and a 6 pfu challenge. However, this study provideslimited benefit for humans or non-human primates because such highdosing is unlikely to be given to humans due to high inherent risks andother factors. So there still exists a need for a human vaccine which isefficacious for protection from Ebola virus infection.

SUMMARY OF THE INVENTION

The present invention satisfies the need discussed above. The presentinvention relates to a method and composition for use in inducing animmune response which is protective against infection with Ebola virus.

Because the biological functions of the individual Ebola virus proteinswere not previously known and the immune mechanisms necessary forpreventing and clearing Ebola virus infection were not previously wellunderstood, it was not known which antigens significantly contribute toprotection and should therefore be included in an eventual vaccinecandidate to induce a protective immune response. However, the inventorshave induced protection against Ebola infection in mammals using virusreplicon particles (VRPs) expressing the Ebola GP, NP, VP24, VP30, VP35or VP40 genes. These VRPs and some uses are described in co-pendingapplication Ser. No. 09/337,946 (filed Jun. 22, 1999), the entirecontents of which are hereby incorporated by reference.

One embodiment of the present invention entails a DNA fragment encodingeach of the Ebola Zaire 1976 GP, NP, VP24, VP30, VP35, and VP40 virionproteins (SEQUENCE ID NOS. 1-7).

Another embodiment provides the DNA fragments of Ebola virion proteinsin a recombinant vector. When the vector is an expression vector, theEbola virion proteins GP, NP, VP24, VP30, VP35, and VP40 are produced.It is preferred that the vector is an alphavirus replicon vector,especially a replicon vector that has the ability to produce the desiredprotein or peptide in a manner that induces protective B and T cells invivo in mammals. Any alphavirus vector may be effective, including butnot limited to the Venezuelan Equine Encephalitis (VEE) virus, easternequine encephalitis, western equine encephalitis, Semliki forest andSindbis. For instance, in a preferred embodiment the VEE replicon vectorcomprises a VEE virus replicon and a DNA fragment encoding any of theEbola Zaire 1976 (Mayinga isolate) GP, NP, VP24, VP30, VP35, or VP40proteins. In another preferred embodiment, the VEE replicon vectorcomprises a VEE virus replicon and a DNA fragment encoding any of theamino acid sequences set forth in SEQ ID NOs:24-53. The construct can beused as a nucleic acid vaccine or for the production of self replicatingRNA. To that end, a self replicating RNA of this invention can comprisethe VEE virus replicon and any of the Ebola Zaire 1976 (Mayinga isolate)RNAs encoding the GP, NP, VP24, VP30, VP35, and VP40 proteins describedabove, or the amino acid sequences set forth in SEQ ID NOs:24-53. TheRNA can be used as a vaccine for protection from Ebola infection. Whenthe RNA is packaged, a VEE virus replicon particle is produced.

Another embodiment entails infectious VEE virus replicon particlesproduced from the VEE virus replicon RNAs described above.

Another embodiment of the invention encompasses peptides that make upcytotoxic T lymphocyte (CTL) epitopes corresponding to Ebola GP, NP,VP24, VP30, VP35, or VP40 proteins. The epitopes may include thesequences identified as SEQ ID NOS:24-53, as described below. A relatedaspect of this embodiment provides DNA fragments that respectivelyencode these Ebola peptides. A further embodiment relates to recombinantDNA constructs that express these epitope peptides.

An additional embodiment includes a pharmaceutical composition thatincludes one or more of these CTL epitope peptides (and preferably oneor more of SEQ ID NOs:24-53), in an effective immunogenic amount in apharmaceutically acceptable carrier and/or adjuvant.

A further embodiment entails an immunological composition for theprotection of mammals including humans against Ebola virus infection,comprising at least one (but preferably at least two, and morepreferably at least three, and most preferably all) of the Ebola virusGP, NP, VP24, VP30, VP35, or VP40 proteins. In a related embodiment, thecomposition may include one or more of the CTL epitopes set forth in SEQID NOs: 24-53 (described below).

In a related preferred embodiment, the immunological compositionscomprise alphavirus replicon particles (such as, for instance, VEE virusreplicon particles) expressing the Ebola virus GP, NP, VP24, VP30, VP35,or VP40 proteins, or any combination of different VEE virus repliconseach expressing one or more different Ebola proteins selected from GP,NP, VP24, VP30, VP35 and VP40. For instance, in a preferred embodimentthe composition may include one or more of SEQ ID NOs: 24-53 (describedbelow). In another preferred embodiment, the composition includes atleast the VP30, VP35 and VP40 proteins.

An additional embodiment includes vaccines against infection by Ebola ,comprising virus replicon particles (preferably VEE virus repliconparticles) expressing the Ebola virus GP, NP, VP24, VP30, VP35, or VP40proteins, or any combination of different VEE virus replicons eachexpressing one or more different Ebola proteins selected from GP, NP,VP24, VP30, VP35 and VP40. For instance, in a preferred embodiment theEbola VRPs contain one or more of the peptides specified by SEQ ID NOs:24-53. In a related embodiment, the vaccine may include at a minimum atleast one of the Ebola proteins selected from GP, NP, VP24, VP30, VP35and VP40. For instance, in a preferred embodiment the vaccine includesat least the VP30, VP35 and VP40 proteins. In another preferredembodiment, the vaccine may include one or more of SEQ ID NOs: 24-53.

The invention also contemplates methods for inducing in a mammal acytotoxic T lymphocyte response to the Ebola virus GP, NP, VP24, VP30,VP35, or VP40 proteins, or to a peptide comprising at least 6 aminoacids thereof. In one version of the method, a recombinant DNA constructis administered to a mammal, such as, for example, a mouse, a guineapig, a monkey or a human, which a recombinant DNA construct expressesthe amino acid sequence of at least one of the Ebola virus GP, NP, VP24,VP30, VP35, or VP40 proteins (or a peptide comprising at least 6 aminoacids thereof), under such conditions that a protective CTL response isinduced in that mammal. In particular, the administered peptides mayinclude one or more of SEQ ID NOs: 24-53. In another version of themethod, one of the above-described immunogenic compositions isadministered to the mammal, and preferably one that comprises virusreplicon particles containing one of the Ebola virus GP, NP, VP24, VP30,VP35, or VP40 proteins (or a peptide comprising at least 6 amino acidsthereof), or including one of the CTL epitopes set forth in SEQ IDNOs:24-53. In another version of the method, the amino acid sequence ofat least one of the Ebola virus GP, NP, VP24, VP30, VP35, or VP40proteins (or a peptide comprising at least 6 amino acids thereof) isadministered to a mammal, such as, for example, a mouse, a guinea pig, amonkey or a human, under such conditions that a protective CTL responseis induced in that mammal. In particular, the administered peptides mayinclude one or more of SEQ ID NOs: 24-53.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood with reference to the followingdescription and appended claims, and accompanying drawings where:

FIG. 1 is a schematic description of the organization of the Ebola virusgenome.

FIGS. 2A, 2B and 2C are schematic representations of the VEE repliconconstructs containing Ebola genes.

FIG. 3 shows the generation of VEE viral-like particles containing Ebolagenes.

FIG. 4 is an immunoprecipitation of Ebola proteins produced fromreplicon constructs.

DETAILED DESCRIPTION

In the description that follows, a number of terms used in recombinantDNA, virology and immunology are extensively utilized. In order toprovide a clearer and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

Filoviruses. The filoviruses (e.g. Ebola Zaire 1976) cause acutehemorrhagic fever characterized by high mortality. Humans can contractfiloviruses by infection in endemic regions, by contact with importedprimates, and by performing scientific research with the virus. However,there currently are no available vaccines or effective therapeutictreatments for filovirus infection in humans. The virions of filovirusescontain seven proteins: a membrane-anchored glycoprotein (GP), anucleoprotein (NP), an RNA-dependent RNA polymerase (L), and four virionstructural proteins (VP24, VP30, VP35, and VP40). Little is known aboutthe biological functions of these proteins and it is not known whichantigens significantly contribute to protection and should therefore beused in an eventual vaccine candidate.

Replicon. A replicon is equivalent to a full-length virus from which allof the viral structural proteins have been deleted. A multiple cloningsite can be inserted downstream of the 26S promoter into the sitepreviously occupied by the structural protein genes. Virtually anyheterologous gene may be inserted into this cloning site. The RNA thatis transcribed from the replicon is capable of replicating andexpressing viral proteins in a manner that is similar to that seen withthe full-length infectious virus clone. However, in lieu of the viralstructural proteins, the heterologous antigen is expressed from the 26Spromoter in the replicon. This system does not yield any progeny virusparticles because there are no viral structural proteins available topackage the RNA into particles.

Particles which appear structurally identical to virus particles can beproduced by supplying structural protein RNAs in trans for packaging ofthe replicon RNA. This is typically done with two defective helper RNAswhich encode the structural proteins. One helper consists of a fulllength infectious clone from which the nonstructural protein genes andthe glycoprotein genes are deleted. This helper retains only theterminal nucleotide sequences, the promoter for subgenomic mRNAtranscription and the sequences for the viral nucleocapsid protein. Thesecond helper is identical to the first except that the nucleocapsidgene is deleted and only the glycoprotein genes are retained. The helperRNAs are transcribed in vitro and are co-transfected with replicon RNA.Because the replicon RNA retains the sequences for packaging by thenucleocapsid protein, and because the helpers lack these sequences, onlythe replicon RNA is packaged by the viral structural proteins. Thepackaged replicon particles are released from the host cell and can thenbe purified and inoculated into animals. The packaged replicon particleswill have a tropism similar to the parent virus. The packaged repliconparticles will infect cells and initiate a single round of replication,resulting in the expression of only the virus nonstructural proteins andthe product of the heterologous gene that was cloned in the place of thevirus structural proteins. In the absence of RNA encoding the virusstructural proteins, no progeny virus particles can be produced from thecells infected by packaged replicon particles.

Any alphavirus replicon may be effective in this invention, as long asit has the ability to produce the desired protein or peptide in a mannerthat induces protective B and T cells in vivo in mammals to which it isadministered to (such as, for instance, eastern equine encephalitis,western equine encephalitis, Semlike forest, Sindbis and VenezualenEquine Encephalitis).

The VEE virus replicon (Vrep) is a preferred vector system. The Vrep isa genetically reorganized version of the VEE virus genome in which thestructural protein genes are replaced with a gene from an immunogen ofinterest, such as the Ebola virus virion proteins. This replicon can betranscribed to produce a self-replicating RNA that can be packaged intoinfectious particles using defective helper RNAs that encode theglycoprotein and capsid proteins of the VEE virus. Since the packagedreplicons do not encode the structural proteins, they are incapable ofspreading to new cells and therefore undergo a single abortive round ofreplication in which large amounts of the inserted immunogen are made inthe infected cells. The VEE virus replicon system is described in U.S.patent to Johnston et al., U.S. Pat. No. 5,792,462 issued on Aug. 11,1998.

Subject. Includes both human, animal, e.g., horse, donkey, pig, mouse,hamster, monkey, chicken, and insect such as mosquito.

In one embodiment, the present invention relates to DNA fragments whichencode any of the Ebola Zaire 1976 (Mayinga isolate) GP, NP, VP24, VP30,VP35, and VP40 proteins. The GP and NP genes of Ebola Zaire werepreviously sequenced by Sanchez et al. (1993, supra) and have beendeposited in GenBank (accession number L11365). A plasmid encoding theVEE replicon vector containing a unique ClaI site downstream from the26S promoter was described previously (Davis, N. L. et al., (1996) J.Virol. 70, 3781-3787; Pushko, P. et al. (1997) Virology 239, 389-401).The Ebola GP and NP genes from the Ebola Zaire 1976 virus were derivedfrom PS64- and PGEM3ZF(−)-based plasmids (Sanchez, A. et al. (1989)Virology 170, 81-91; Sanchez, A. et al. (1993) Virus Res. 29, 215-240).From these plasmids, the BamHI-EcoRI (2.3 kb) and BamHI-KpnI (2.4 kb)fragments containing the NP and GP genes, respectively, were subclonedinto a shuttle vector that had been digested with BamHI and EcoRI (Daviset al. (1996) supra; Grieder, F. B. et al. (1995) Virology 206,994-1006). For cloning of the GP gene, overhanging ends produced by KpnI(in the GP fragment) and EcoRI (in the shuttle vector) were made bluntby incubation with T4 DNA polymerase according to methods known in theart. From the shuttle vector, GP or NP genes were subcloned asClaI-fragments into the ClaI site of the replicon clone, resulting inplasmids encoding the GP or NP genes in place of the VEE structuralprotein genes downstream from the VEE 26S promoter.

The VP genes of Ebola Zaire were previously sequenced by Sanchez et al.(1993, supra) and have been deposited in GenBank (accession numberL11365). The VP genes of Ebola used in the present invention were clonedby reverse transcription of RNA from Ebola-infected Vero E6 cells andsubsequent amplification of viral cDNAs using the polymerase chainreaction. First strand synthesis was primed with oligo dT (LifeTechnologies). Second strand synthesis and subsequent amplification ofviral cDNAs were performed with gene-specific primers (SEQ ID NOS:8-16).The primer sequences were derived from the GenBank deposited sequencesand were designed to contain a ClaI restriction site for cloning theamplified VP genes into the ClaI site of the replicon vector. Theletters and numbers in bold print indicate Ebola gene sequences in theprimers and the corresponding location numbers based on the GenBankdeposited sequences.

VP24: (1) forward primer is (SEQ ID NO:8)5′-GGGATCGATCTCCAGACACCAAGCAAGACC-3′         (10,311-10,331)       (2)reverse primer is (SEQ ID NO:9) 5′-GGGATCGATGAGTCAGCATATATGAGTTAGCTC-3′      (11,122-11,145) VP30: (1) forward primer is SEQ ID NO:10)5′-CCCATCGATCAGATCTGCGAACCGGTAGAG-3′      (8408-8430)       (2) reverseprimer is (SEQ ID NO:11) 5′-CCCATCGATGTACCCTCATCAGACCATGAGC-3′        (9347-9368) VP35: (1) forward primer is (SEQ ID NO:12)5′-GGGATCGATAGAAAAGCTGGTCTAACAAGATGA-3′     (3110-3133)       (2)reverse primer is (SEQ ID NO:13)5′-CCCATCGATCTCACAAGTGTATCATTAATGTAACGT-3′         (4218-4244) VP40: (1)forward primer is (SEQ ID NO:14) 5′-CCCATCGATCCTACCTCGGCTGAGAGAGTG-3′           (4408-4428)       (2) reverse primer is (SEQ ID NO:15)5′-CCCATCGATATGTTATGCACTATCCCTGAGAAG-3′            (5495-5518) VP30 #2:      (1) forward primer as for VP30 above       (2) reverse primer is(SEQ ID NO:16) 5′-CCCATCGATCTGTTAGGGTTGTATCATACC-3′

The Ebola virus genes cloned into the VEE replicon were sequenced.Changes in the DNA sequence relative to the sequence published bySanchez et al. (1993) are described relative to the nucleotide (nt)sequence number from GenBank (accession number L11365).

The nucleotide sequence we obtained for Ebola virus GP (SEQ ID NO:1)differed from the GenBank sequence by a transition from A to G at nt8023. This resulted in a change in the amino acid sequence from Ile toVal at position 662 (SEQ ID NO: 17).

The nucleotide sequence we obtained for Ebola virus NP (SEQ ID NO:2)differed from the GenBank sequence at the following 4 positions:insertion of a C residue between nt 973 and 974, deletion of a G residueat nt 979, transition from C to T at nt 1307, and a transversion from Ato C at nt 2745. These changes resulted in a change in the proteinsequence from Arg to Glu at position 170 and a change from Leu to Phe atposition 280 (SEQ ID NO: 18).

The Ebola virus VP24 nucleotide sequence (SEQ ID NO:3) differed from theGenBank sequence at 6 positions, resulting in 3 nonconservative changesin the amino acid sequence. The changes in the DNA sequence of VP24consisted of a transversion from G to C at nt 10795, a transversion fromC to G at nt 10796, a transversion from T to A at nt 10846, atransversion from A to T at nt 10847, a transversion from C to G at nt11040, and a transversion from C to G at nt 11041. The changes in theamino acid sequence of VP24 consisted of a Cys to Ser change at position151, a Leu to His change at position 168, and a Pro to Gly change atposition 233 (SEQ ID NO: 19).

Two different sequences for the Ebola virus VP30 gene, VP30 and VP30#2(SEQ ID NOS: 4 and 7) are included. Both of these sequences differ fromthe GenBank sequence by the insertion of an A residue in the upstreamnoncoding sequence between nt 8469 and 8470 and an insertion of a Tresidue between nt 9275 and 9276 that results in a change in the openreading frame of VP30 and VP30#2 after position 255 (SEQ ID NOS: 20 and23). As a result, the C-terminus of the VP30 protein differssignificantly from that previously reported. In addition to these 2changes, the VP30#2 nucleic acid in SEQ ID NO:7 contains a conservativetransition from T to C at nt 9217. Because the primers originally usedto clone the VP30 gene into the replicon were designed based on theGenBank sequence, the first clone that we constructed (SEQ ID NO: 4) didnot contain what we believe to be the authentic C-terminus of theprotein. Therefore, in the absence of the VP30 stop codon, theC-terminal codon was replaced with 37 amino acids derived from thevector sequence. The resulting VP30 construct therefore differed fromthe GenBank sequence in that it contained 32 amino acids of VP30sequence (positions 256 to 287, SEQ ID NO:20) and 37 amino acids ofirrelevant sequence (positions 288 to 324, SEQ ID NO:20) in the place ofthe C-terminal 5 amino acids reported in GenBank. However, inclusion of37 amino acids of vector sequence in place of the C-terminal amino acid(Pro, SEQ ID NO: 23) did not inhibit the ability of the protein to serveas a protective antigen in BALB/c mice. We have also determined that aVEE replicon construct, which contains the authentic C-terminus of VP30(VP30#2, SEQ ID NO: 23), protects mice against a lethal Ebola challenge.

The nucleotide sequence for Ebola virus VP35 (SEQ ID NO:5) differed fromthe GenBank sequence by a transition from T to C at nt 4006, atransition from T to C at nt 4025, and an insertion of a T residuebetween nt 4102 and 4103. These sequence changes resulted in a changefrom a Ser to a Pro at position 293 and a change from Phe to Ser atposition 299 (SEQ ID NO: 21). The insertion of the T residue resulted ina change in the open reading frame of VP35 from that previously reportedby Sanchez et al. (1993) following amino acid number 324. As a result,Ebola virus VP35 encodes a protein of 340 amino acids, where amino acids325 to 340 (SEQ ID NO: 21) differ from and replace the C-terminal 27amino acids of the previously published sequence.

Sequencing of VP30 and VP35 was also performed on RT/PCR products fromRNA derived from cells that were infected with Ebola virus 1976, Ebolavirus 1995 or the mouse-adapted Ebola virus. The changes noted above forthe Vrep constructs were also found in these Ebola viruses. Thus, webelieve that these changes are real events and not artifacts of cloning.

The Ebola virus VP40 nucleotide sequence (SEQ ID NO:6) differed from theGenBank sequence by a transversion from a C to G at nt 4451 and atransition from a G to A at nt 5081. These sequence changes did notalter the protein sequence of VP40 (SEQ ID NO: 22) from that of thepublished sequence.

Each of the Ebola virus genes were individually inserted into a VEEvirus replicon vector. The VP24, VP30, VP35, and VP40 genes of EbolaZaire 1976 (Mayinga isolate) were cloned by reverse transcription of RNAfrom Ebola-infected Vero E6 cells and viral cDNAs were amplified usingthe polymerase chain reaction. The Ebola Zaire 1976 (Mayinga isolate) GPand NP genes were obtained from plasmids already containing these genes(Sanchez, A. et al., (1989) Virology 170, 81-91; Sanchez, A. etal.,(1993) Virus Res. 29, 215-240) and were subcloned into the VEEreplicon vector.

After characterization of the Ebola gene products expressed from the VEEreplicon constructs in cell culture, these constructs were packaged intoinfectious VEE virus replicon particles (VRPs) and subcutaneouslyinjected into BALB/c and C57BL/6 mice. As controls in these experiments,mice were also immunized with a VEE replicon expressing Lassanucleoprotein (NP) as an irrelevant control antigen, or injected withPBS buffer alone. The results of this study demonstrate that VRPsexpressing the Ebola GP, NP, VP24, VP30, VP35 or VP40 genes inducedprotection in mice and may reasonably to expected to provide protectionin humans.

DNA or polynucleotide sequences to which the invention also relatesinclude sequences of at least about 6 nucleotides, preferably at leastabout 8 nucleotides, more preferably at least about 10-12 nucleotides,most preferably at least about 15-20 nucleotides corresponding, i.e.,homologous to or complementary to, a region of the Ebola nucleotidesequences described above. Preferably, the sequence of the region fromwhich the polynucleotide is derived is homologous to or complementary toa sequence which is unique to the Ebola genes. Whether or not a sequenceis unique to the Ebola gene can be determined by techniques known tothose of skill in the art. For example, the sequence can be compared tosequences in databanks, e.g., GenBank and compared by DNA:DNAhybridization. Regions from which typical DNA sequences may be derivedinclude but are not limited to, for example, regions encoding specificepitopes, as well as non-transcribed and/or non-translated regions.

The derived polynucleotide is not necessarily physically derived fromthe nucleotide sequences shown in SEQ ID NO:1-7, but may be generated inany manner, including for example, chemical synthesis or DNA replicationor reverse transcription or transcription, which are based on theinformation provided by the sequence of bases in the region(s) fromwhich the polynucleotide is derived. In addition, combinations ofregions corresponding to that of the designated sequence may be modifiedin ways known in the art to be consistent with an intended use. Thesequences of the present invention can be used in diagnostic assays suchas hybridization assays and polymerase chain reaction assays, forexample, for the discovery of other Ebola sequences.

In another embodiment, the present invention relates to a recombinantDNA molecule that includes a vector and a DNA sequence as describedabove. The vector can take the form of a plasmid, a eukaryoticexpression vector such as pcDNA3.1, pRcCMV2, pZeoSV2,or pCDM8, which areavailable from Invitrogen, or a virus vector such as baculovirusvectors, retrovirus vectors or adenovirus vectors, alphavirus vectors,and others known in the art.

In a further embodiment, the present invention relates to host cellsstably transformed or transfected with the above-described recombinantDNA constructs. The host cell can be prokaryotic (for example,bacterial), lower eukaryotic (for example, yeast or insect) or highereukaryotic (for example, all mammals, including but not limited to mouseand human). Both prokaryotic and eukaryotic host cells may be used forexpression of the desired coding sequences when appropriate controlsequences which are compatible with the designated host are used.

Among prokaryotic hosts, E. coli is the most frequently used host cellfor expression. General control sequences for prokaryotes includepromoters and ribosome binding sites. Transfer vectors compatible withprokaryotic hosts are commonly derived from a plasmid containing genesconferring ampicillin and tetracycline resistance (for example, pBR322)or from the various pUC vectors, which also contain sequences conferringantibiotic resistance. These antibiotic resistance genes may be used toobtain successful transformants by selection on medium containing theappropriate antibiotics. Please see e.g., Maniatis, Fitsch and Sambrook,Molecular Cloning; A Laboratory Manual (1982) or DNA Cloning, Volumes Iand II (D. N. Glover ed. 1985) for general cloning methods. The DNAsequence can be present in the vector operably linked to sequencesencoding an IgG molecule, an adjuvant, a carrier, or an agent for aid inpurification of Ebola proteins, such as glutathione S-transferase.

In addition, the Ebola virus gene products can also be expressed ineukaryotic host cells such as yeast cells and mammalian cells.Saccharomyces cerevisiae, Saccharomyces carlsbergensis, and Pichiapastoris are the most commonly used yeast hosts. Control sequences foryeast vectors are known in the art. Mammalian cell lines available ashosts for expression of cloned genes are known in the art and includemany immortalized cell lines available from the American Type CultureCollection (ATCC), such as CHO cells, Vero cells, baby hamster kidney(BHK) cells and COS cells, to name a few. Suitable promoters are alsoknown in the art and include viral promoters such as that from SV40,Rous sarcoma virus (RSV), adenovirus (ADV), bovine papilloma virus(BPV), and cytomegalovirus (CMV). Mammalian cells may also requireterminator sequences, poly A addition sequences, enhancer sequenceswhich increase expression, or sequences which cause amplification of thegene. These sequences are known in the art.

The transformed or transfected host cells can be used as a source of DNAsequences described above. When the recombinant molecule takes the formof an expression system, the transformed or transfected cells can beused as a source of the protein described below.

In another embodiment, the present invention relates to Ebola virionproteins such as

GP having an amino acid sequence corresponding to SEQ ID NO:17encompassing 676 amino acids,

NP, having an amino acid sequence corresponding to SEQ ID NO:18encompassing 739 amino acids,

VP24, having an amino acid sequence corresponding to SEQ ID NO:19encompassing 251 amino acids,

VP30, having an amino acid sequence corresponding SEQ ID NO:20encompassing 324 amino acids,

VP35, having an amino acid sequence corresponding to SEQ ID NO:21encompassing 340 amino acids, and

VP40, having an amino acid sequence corresponding to SEQ ID NO:22,encompassing 326 amino acids, and

VP30#2, having an amino acid sequence corresponding to SEQ ID NO:23encompassing 288 amino acids, or any allelic variation of these aminoacid sequences. By allelic variation is meant a natural or syntheticchange in one or more amino acids which occurs between differentserotypes or strains of Ebola virus and does not affect the antigenicproperties of the protein. There are different strains of Ebola (Zaire1976, Zaire 1995, Reston, Sudan, and Ivory Coast). The NP and VP genesof all these different viruses have not been sequenced. It would beexpected that these proteins would have homology among different strainsand that vaccination against one Ebola virus strain might afford crossprotection to other Ebola virus strains.

A polypeptide or amino acid sequence derived from any of the amino acidsequences in SEQ ID NO:17, 18, 19, 20, 21, 22, and 23 refers to apolypeptide having an amino acid sequence identical to that of apolypeptide encoded in the sequence, or a portion thereof wherein theportion consists of at least 2-5 amino acids, preferably at least 8-10amino acids, and more preferably at least 11-15 amino acids, or which isimmunologically identifiable with a polypeptide encoded in the sequence.

A recombinant or derived polypeptide is not necessarily translated froma designated nucleic acid sequence, or the DNA sequence found in GenBankaccession number L11365. It may be generated in any manner, includingfor example, chemical synthesis, or expression from a recombinantexpression system.

When the DNA or RNA sequences described above are in a repliconexpression system, such as the VEE replicon described above, theproteins can be expressed in vivo. The DNA sequence for any of the GP,NP, VP24, VP30, VP35, and VP40 virion proteins can be cloned into themultiple cloning site of a replicon such that transcription of the RNAfrom the replicon yields an infectious RNA encoding the Ebola protein orproteins of interest (see FIGS. 2A, 2B and 2C). The replicon constructsinclude Ebola virus GP (SEQ ID NO:1) cloned into a VEE replicon(VRepEboGP), Ebola virus NP (SEQ ID NO:2) cloned into a VEE replicon(VRepEboNP), Ebola virus VP24 (SEQ ID NO:3) cloned into a VEE replicon(VRepEboVP24), Ebola virus VP30 (SEQ ID NO:4) or VP30#2 (SEQ ID NO:7)cloned into a VEE replicon (VRepEboVP30 or VRepEboVP30(#2)), Ebola virusVP35 (SEQ ID NO:5) cloned into a VEE replicon (VRepEboVP35), and Ebolavirus VP40 (SEQ ID NO:6) cloned into a VEE replicon (VRepEboVP40). Thereplicon DNA or RNA can be used as a vaccine for inducing protectionagainst infection with Ebola.

Use of helper RNAs containing sequences necessary for packaging of theviral replicon transcripts will result in the production of virus-likeparticles containing replicon RNAs (FIG. 3). These packaged repliconswill infect host cells and initiate a single round of replicationresulting in the expression of the Ebola proteins in the infected cells.The packaged replicon constructs (i.e. VEE virus replicon particles,VRP) include those that express Ebola virus GP (EboGPVRP), Ebola virusNP (EboNPVRP), Ebola virus VP24 (EboVP24VRP), Ebola virus VP30(EboVP30VRP or EboVP30VRP(#2)), Ebola virus VP35 (EboVP35VRP), and Ebolavirus VP40 (EboVP40VRP).

In another embodiment, the present invention relates to RNA moleculesresulting from the transcription of the constructs described above. TheRNA molecules can be prepared by in vitro transcription using methodsknown in the art and described in the Examples below. Alternatively, theRNA molecules can be produced by transcription of the constructs invivo, and isolating the RNA. These and other methods for obtaining RNAtranscripts of the constructs are known in the art. Please see CurrentProtocols in Molecular Biology. Frederick M. Ausubel et al. (eds.), JohnWiley and Sons, Inc. The RNA molecules can be used, for example, as adirect RNA vaccine, or to transfect cells along with RNA from helperplasmids, one of which expresses VEE glycoproteins and the other VEEcapsid proteins, as described above, in order to obtain repliconparticles.

In a further embodiment, the present invention relates to a method ofproducing the recombinant or fusion protein which includes culturing theabove-described host cells under conditions such that the DNA fragmentis expressed and the recombinant or fusion protein is produced thereby.The recombinant or fusion protein can then be isolated using methodologywell known in the art. The recombinant or fusion protein can be used asa vaccine for immunity against infection with Ebola or as a diagnostictool for detection of Ebola infection.

In another embodiment, the present invention relates to antibodiesspecific for the above-described recombinant proteins (or polypeptides).For instance, an antibody can be raised against a peptide having theamino acid sequence of any of SEQ ID NO:17-25, or against a portionthereof of at least 10 amino acids, preferably, 11-15 amino acids.Persons with ordinary skill in the art using standard methodology canraise monoclonal and polyclonal antibodies to the protein(orpolypeptide) of the present invention, or a unique portion thereof.Materials and methods for producing antibodies are well known in the art(see for example Goding, In Monoclonal Antibodies: Principles andPractice, Chapter 4, 1986).

In another embodiment, the present invention relates to an Ebola vaccinecomprising VRPs that express one or more of the Ebola proteins describedabove. The vaccine is administered to a subject wherein the replicon isable to initiate one round of replication producing the Ebola proteinsto which a protective immune response is initiated in said subject.

It is likely that the protection afforded by these genes is due to boththe humoral (antibodies (Abs)) and cellular (cytotoxic T cells (CTLs))arms of the immune system. Protective immunity induced to a specificprotein may comprise humoral immunity, cellular immunity, or both. Theonly Ebola virus protein known to be on the outside of the virion is theGP. The presence of GP on the virion surface makes it a likely targetfor GP-specific Abs that may bind either extracellular virions orinfected cells expressing GP on their surfaces. Serum transfer studiesin this invention demonstrate that Abs that recognize GP protect miceagainst lethal Ebola virus challenge.

In contrast, transfer of Abs specific for NP, VP24, VP30, VP35, or VP40did not protect mice against lethal Ebola challenge. This data, togetherwith the fact that these are internal virion proteins that are notreadily accessible to Abs on either extracellular virions or the surfaceof infected cells, suggest that the protection induced in mice by theseproteins is mediated by CTLs.

CTLs can bind to and lyse virally infected cells. This process beginswhen the proteins produced by cells are routinely digested intopeptides. Some of these peptides are bound by the class I or class IImolecules of the major histocompatability complex (MHC), which are thentransported to the cell surface. During virus infections, viral proteinsproduced within infected cells also undergo this process. CTLs that havereceptors that bind to both a specific peptide and the MHC moleculeholding the peptide lyse the peptide-bearing cell, thereby limitingvirus replication. Thus, CTLs are characterized as being specific for aparticular peptide and restricted to a class I or class II MHC molecule.

CTLs may be induced against any of the Ebola virus proteins, as all ofthe viral proteins are produced and digested within the infected cell.Thus, protection to Ebola virus involves CTLs against GP, NP, VP24,VP30, VP35, and/or VP40. It is especially noteworthy that the VPproteins varied in their protective efficacy when tested in geneticallyinbred mice that differ at the MHC locus. This, together with theinability to demonstrate a role for Abs in protection induced by the VPproteins and the data in Table A below, demonstrates a role for CTLs.Thus, in this invention a vaccine may include several Ebola virusproteins (e.g., at least two), or several CTL epitopes (e.g., at leasttwo), capable of inducing broad protection to different Ebola viruses inoutbred populations (e.g. people). To that end, the inventors haveidentified 18 sequences recognized by CTLs, as determined initially bymeasuring gamma interferon production by intracellular cytokine stainingand gamma interferon secretion by the ELISpot assay. The ability to lysecells was measured in chromium release assays and protection wasevaluated by adoptive transfer of cells into Ebola-naïve mice. Wheresequence information is available, the conservation of these CTLepitopes in other Ebola viruses is noted in Table A. Conserved sequencesshould be capable of inducing protective CTLs to each of the viruses inwhich the sequence is present.

The identified CTL epitopes are:

-   -   Ebola virus NP SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, and SEQ        ID NO:28;    -   Ebola virus GP SEQ ID NO:29 (encompassing YFGPAAEGI, SEQ ID        NO:42);    -   Ebola virus VP24 SEQ ID NO:25 (encompassing KFINKLDAL, SEQ ID        NO:43), SEQ ID NO:30 (encompassing NYNGLLSSI, SEQ ID NO:44), and        SEQ ID NO:31 (encompassing PGPAKFSLL, SEQ ID NO:45);    -   Ebola virus VP30 SEQ ID NO:32 (encompassing LSLLCETHLR, SEQ ID        NO:46), and SEQ ID NO:33 (encompassing MFITAFLNI, SEQ ID NO:47);    -   Ebola virus VP35 SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36;        and    -   Ebola virus VP40 SEQ ID NO:37 (encompassing EFVLPPVQL, SEQ ID        NO:48), SEQ ID NO:38 (encompassing FLVPPV, SEQ ID NO:49 and        QYFTFDLTALK, SEQ ID NO:50), SEQ ID NO:39 (encompassing        TSPEKIQAI, SEQ ID NO:51); SEQ ID NO:40 (encompassing RIGNQAFL,        SEQ ID NO:52), and SEQ ID NO:41 (encompassing QAFLQEFV, SEQ ID        NO:53).        See Table A below.

Testing to identify the role of CTLs in protection was performed byobtaining CTLs from mice, expanding them in vitro, and transferring thecells into an unvaccinated mouse of the same genetic background. Severalhours later, the recipient mice were challenged with 10-1000 pfu ofmouse-adapted Ebola virus. They were observed for signs of illness for28 days. Control animals received no cells or cells that were notspecific for Ebola virus. Testing of the cells in vitro indicated thatthey were CD8+, a marker indicating class I-restriction. The inventorswere able to demonstrate that CTLs to these sequences protected at least80% of recipient mice from challenge. In all of our examples, theability to lyse peptide-pulsed target cells has predicted protection inmice receiving those cells.

TABLE A Ebola Virus Epitopes Recognized by Murine CD8 + T cells % INF-γINF-γ Conserved Protein^(a) Epitope^(b) ICC^(c) ELISpot^(d) ⁵¹Cr^(e)Protective^(f) Restriction^(g) Strains^(h) GP WIPYFGPAAEGIYTE (SEQ ID29) 0.40/0.08 Y Neg IP H-2^(b) R,G NP VYQVNNLEEIC (SEQ ID 24) 1.06/0.11Y 55.6 Yes H-2^(b) G GQFLFASL (SEQ ID 26) 0.88/0.11 Y 45   Yes H-2^(b)S,G DAVLYYHMM (SEQ ID 27) 0.99/0.11 Y 40.6 Yes H-2^(b) G SFKAALSSL (SEQID 28) 0.63/0.04 Y 38.8 Yes H-2^(d) VP24 NILKFINKLDALHVV (SEQ ID 25)0.52/0.09 Y 45.9 Yes H-2^(d) G NYNGLLSSIEGTQN (SEQ ID 30) 0.38/0.09 Y50.6 Yes H-2^(d) R,G RMKPGPAKFSLLHESTLKAFTQGSS 3.34/0.09 Y 43.2 Yes*H-2^(d) R,G (SEQ ID 31) VP30 FSKSQLSLLCETHLR (SEQ ID 32) 0.45/0.15 N47.3 Yes* H-2^(b) DLQSLIMFITAFLNI (SEQ ID 33) 0.7/0.15 N ND Yes* H-2^(b)VP35 RNIMYDHL (SEQ ID 34) 1.53/0.22 Y 87.4 Yes H-2^(b) MVAKYDLL (SEQ ID35) 1.63/0.22 N 78.9 Yes H-2^(b) R CDIENNPGL (SEQ ID 36) 1.99/0.15 N80.4 Yes H-2^(b) VP40 AFLQEFVLPPVQLPQ (SEQ ID 37) 0.45/0.22 ND ND IPH-2^(d) FVLPPVQLPQYFTFDLTALK (SEQ ID 38) 0.41/0.22 ND 38   Yes* H-2^(d)KSGKKGNSADLTSPEKIQAIMTSLQDFKIV 0.6/0.22 N 36.4 IP H-2^(d) (SEQ ID 39)PLRLLRIGNQAFLQE (SEQ ID 40) 0.7/0.05 N 52.8 Yes H-2^(b) RIGNQAFLQEFVLPP(SEQ ID 41) 0.38/0.05 N 46.7 IP H-2^(b) ^(a)Proteins are from EbolaZaire '76 virus: GP, glycoprotein, NP, nucleoprotein, or the virionproteins VP24, VP30, VP35 or VP40. ^(b)Epitope, indicates peptidesequence(s) tested in the T cell assays. Underlined regions are presumedminimum epitopes based on binding motifs, algorithm predictions and/ordemonstrated effects based on synthesis of shorter peptides. ^(c)ICCdata is % of CD8 that are INF-γ positive and CD8 positive/background^(d)IFN-γ ELISpot assays indicated (Y, yes; N, no) presence of secretedinterferon-γ. ^(e51)Cr data is specific lysis at the 25:1 E:T ratio.^(f)Protection observed in 100% of naive mice receiving CTLs specificfor the designated epitopes, except where marked with *, in which casesprotection of 80–90% was observed. IP, in progress (data within 2weeks). ^(g)Restriction indicates the major histocompatability type forwhich lysis was observed. H-2^(b) mice are C57B1/6 and H-2^(d)areBalb/c. ^(h)Conserved strains: underlined sequences representingepitopes are identical in Sudan, Gabon and Reston Ebola viruses asindicated by S, G or R, respectively. ND, not determined

In another embodiment, the invention relates to a vaccine against Ebolainfection including at least one of these CTL epitope sequences, andpreferably at least one CTL epitope having the amino acid sequence ofSEQ ID NOs:24-53. Preferably, the vaccine includes at least two of theCTL epitope sequences, more preferably at least three, more preferablyat least four, more preferably at least five, and more preferably all ofthe sequences. As shown in the examples below, protection is increasedas the number of CTL epitopes in the immunogenic composition or vaccineis increased, and also as the number of epitopes from different Ebolaproteins is increased.

In another embodiment, the vaccine includes a CTL epitope sequence fromat least two different proteins selected from the group consisting ofGP, NP, VP24, VP30, VP35 and VP40. More preferably, the vaccine includesa CTL epitope sequence from at least three different proteins from thatgroup, more preferably at least four, more preferably at least five, andmost preferably includes at least one CTL epitope sequence from each ofthe six proteins. The CTL epitopes may have the amino acid sequences asset forth in SEQ ID NOs:24-53. It is noted that administering the GPpeptide alone may prevent the induction of protective antibodies, whichmay be undesirable.

In a further vaccine embodiment, the vaccine comprises virus repliconparticles (preferably VEE virus replicon particles but other alphavirusreplicon particles will do as described above) expressing the Ebolavirus GP, NP, VP24, VP30, VP35, or VP40 proteins, or any combination ofdifferent VEE virus replicons each expressing one or more differentEbola proteins selected from GP, NP, VP24, VP30, VP35 and VP40. Forinstance, in a preferred embodiment the Ebola VRPs express one or moreof the peptides specified by SEQ ID NOs: 24-53.

In another vaccine embodiment, the vaccine may include at a minimum atleast one of the Ebola proteins selected from GP, NP, VP24, VP30, VP35and VP40, but preferably contains at least two, more preferably at leastthree, more preferably at least four, more preferably at least five, andmost preferably all of them. It is noted again that administering the GPpeptide alone may prevent the induction of protective antibodies, whichmay be undesirable. For instance, in a preferred embodiment the vaccineincludes at least the VP30, VP35 and VP40 proteins. In another preferredembodiment, the vaccine may include one or more of SEQ ID NOs: 24-53.

When considering which type of vaccine may be most effective for anindividual, it is noted that the same protective response could beinduced by the peptide or the full protein produced from the VRPs.Production of the peptide intracellularly is generally preferred becauseit is usually (but not always) more effective than providing itextracellularly. Thus, vaccines containing VRPs may be preferred becausethe VRPs infect cells and therefore achieve intracellular production.

Such vaccines might be delivered as synthetic peptides, or as fusionproteins, alone or co-administered with cytokines and/or adjuvants orcarriers safe for human use, e.g. aluminum hydroxide, to increaseimmunogenicity. In addition, sequences such as ubiquitin can be added toincrease antigen processing for more effective CTL responses.

In yet another embodiment, the present invention relates to a method forproviding immunity against Ebola virus, said method comprisingadministering one or more VRPs expressing any combination of the GP, NP,VP24, VP30 or VP30#2, VP35 and VP40 Ebola proteins to a subject suchthat a protective immune reaction is generated. In another relatedembodiment, the method may entail administering one or more VRPsexpressing any combination of the peptides designated SEQ ID NOs:24-53,or simply one or more of the peptides designated SEQ ID NOs:24-53.

Vaccine formulations of the present invention comprise an immunogenicamount of a VRP, such as for example EboVP24VRP described above, or, fora multivalent vaccine, a combination of replicons, in a pharmaceuticallyacceptable carrier. An “immunogenic amount” is an amount of the VRP(s)sufficient to evoke an immune response in the subject to which thevaccine is administered. An amount of from about 10⁴-10⁸ focus-formingunits per dose is suitable, depending upon the age and species of thesubject being treated. The subject may be inoculated 2-3 times.Exemplary pharmaceutically acceptable carriers include, but are notlimited to, sterile pyrogen-free water and sterile pyrogen-freephysiological saline solution.

Administration of the VRPs disclosed herein may be carried out by anysuitable means, including parenteral injection (such as intraperitoneal,subcutaneous, or intramuscular injection), in ovo injection of birds,orally, or by topical application of the virus (typically carried in apharmaceutical formulation) to an airway surface. Topical application ofthe virus to an airway surface can be carried out by intranasaladministration (e.g., by use of dropper, swab, or inhaler which depositsa pharmaceutical formulation intranasally). Topical application of thevirus to an airway surface can also be carried out by inhalationadministration, such as by creating respirable particles of apharmaceutical formulation (including both solid particles and liquidparticles) containing the replicon as an aerosol suspension, and thencausing the subject to inhale the respirable particles. Methods andapparatus for administering respirable particles of pharmaceuticalformulations are well known, and any conventional technique can beemployed. Oral administration may be in the form of an ingestable liquidor solid formulation.

When the replicon RNA or DNA is used as a vaccine, the replicon RNA orDNA can be administered directly using techniques such as delivery ongold beads (gene gun), delivery by liposomes, or direct injection, amongother methods known to people in the art. Any one or more DNA constructsor replicating RNA described above can be use in any combinationeffective to elicit an immunogenic response in a subject. Generally, thenucleic acid vaccine administered may be in an amount of about 1-5 ug ofnucleic acid per dose and will depend on the subject to be treated,capacity of the subject's immune system to develop the desired immuneresponse, and the degree of protection desired. Precise amounts of thevaccine to be administered may depend on the judgement of thepractitioner and may be peculiar to each subject and antigen.

The vaccine may be given in a single dose schedule, or preferably amultiple dose schedule in which a primary course of vaccination may bewith 1-10 separate doses, followed by other doses given at subsequenttime intervals required to maintain and or reinforce the immuneresponse, for example, at 1-4 months for a second dose, and if needed, asubsequent dose(s) after several months. Examples of suitableimmunization schedules include: (i) 0, 1 months and 6 months, (ii) 0, 7days and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, (v) 0, 1 and2 months, or other schedules sufficient to elicit the desired immuneresponses expected to confer protective immunity, or reduce diseasesymptoms, or reduce severity of disease.

In a further embodiment, the present invention relates to a method ofdetecting the presence of antibodies against Ebola virus in a sample.Using standard methodology well known in the art, a diagnostic assay canbe constructed by coating on a surface (i.e. a solid support forexample, a microtitration plate, a membrane (e.g. nitrocellulosemembrane) or a dipstick), all or a unique portion of any of the Ebolaproteins described above or any combination thereof, and contacting itwith the serum of a person or animal suspected of having Ebola. Thepresence of a resulting complex formed between the Ebola protein(s) andserum antibodies specific therefor can be detected by any of the knownmethods common in the art, such as fluorescent antibody spectroscopy orcolorimetry. This method of detection can be used, for example, for thediagnosis of Ebola infection and for determining the degree to which anindividual has developed virus-specific antibodies after administrationof a vaccine.

In yet another embodiment, the present invention relates to a method fordetecting the presence of Ebola virion proteins in a sample. Antibodiesagainst GP, NP, and the VP proteins could be used for diagnostic assays.Using standard methodology well known in the art, a diagnostics assaycan be constructed by coating on a surface (i.e. a solid support, forexample, a microtitration plate or a membrane (e.g. nitrocellulosemembrane)), antibodies specific for any of the Ebola proteins describedabove, and contacting it with serum or a tissue sample of a personsuspected of having Ebola infection. The presence of a resulting complexformed between the protein or proteins in the serum and antibodiesspecific therefor can be detected by any of the known methods common inthe art, such as fluorescent antibody spectroscopy or colorimetry. Thismethod of detection can be used, for example, for the diagnosis of Ebolavirus infection.

In another embodiment, the present invention relates to a diagnostic kitwhich contains any combination of the Ebola proteins described above andancillary reagents that are well known in the art and that are suitablefor use in detecting the presence of antibodies to Ebola in serum or atissue sample. Tissue samples contemplated can be from monkeys, humans,or other mammals.

In yet another embodiment, the present invention relates to DNA ornucleotide sequences for use in detecting the presence of Ebola virususing the reverse transcription-polymerase chain reaction (RT-PCR). TheDNA sequence of the present invention can be used to design primerswhich specifically bind to the viral RNA for the purpose of detectingthe presence of Ebola virus or for measuring the amount of Ebola virusin a sample. The primers can be any length ranging from 7 to 400nucleotides, preferably at least 10 to 15 nucleotides, or morepreferably 18 to 40 nucleotides. Reagents and controls necessary for PCRreactions are well known in the art. The amplified products can then beanalyzed for the presence of viral sequences, for example by gelfractionation, with or without hybridization, by radiochemistry, andimmunochemistry techniques.

In yet another embodiment, the present invention relates to a diagnostickit which contains PCR primers specific for Ebola virus and ancillaryreagents for use in detecting the presence or absence of Ebola in asample using PCR. Samples contemplated can be obtained from human,animal, e.g., horse, donkey, pig, mouse, hamster, monkey, or othermammals, birds, and insects, such as mosquitoes.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors and thought to function well inthe practice of the invention, and thus can be considered to constitutepreferred modes for its practice. However, those of skill in the artshould, in light of the present disclosure, appreciate that many changescan be made in the specific embodiments which are disclosed and stillobtain a like or similar result without departing from the spirit andscope of the invention.

The following MATERIALS AND METHODS were used in the examples thatfollow.

Cells Lines and Viruses

BHK (ATCC CCL 10), Vero 76 (ATCC CRL 1587), and Vero E6 (ATCC CRL 1586)cell lines were maintained in minimal essential medium with Earle'ssalts, 5-10% fetal bovine serum, and 50 □g/mL gentamicin sulfate. ForCTL assays, EL4 (ATCC TIB39), L5178Y (ATCC CRL 1723) and P815 (ATCCTIB64) were maintained in Dulbecco's minimal essential mediumsupplemented with 5-10% fetal bovine serum and antibiotics.

A stock of the Zaire strain of Ebola virus originally isolated from apatient in the 1976 outbreak (Mayinga) and passaged intracerebrally 3times in suckling mice and 2 times in Vero cells was adapted to adultmice through serial passage in progressively older suckling mice (Brayet al.,(1998) J. Infect. Dis. 178, 651-661). A plaque-purifiedninth-mouse-passage isolate which was uniformly lethal for adult mice(“mouse-adapted virus”) was propagated in Vero E6 cells, aliquotted, andused in all mouse challenge experiments and neutralization assays.

A stock of the Zaire strain of Ebola 1976 virus was passaged spleen tospleen in strain 13 guinea pigs four times. This guinea pig-adaptedstrain was used to challenge guinea pigs.

Construction and Packaging of Recombinant VEE Virus Replicons (VRPs)

Replicon RNAs were packaged into VRPs as described (Pushko et al., 1997,supra). Briefly, capped replicon RNAs were produced in vitro by T7run-off transcription of NotI-digested plasmid templates using theRiboMAX T7 RNA polymerase kit (Promega). BHK cells were co-transfectedwith the replicon RNAs and the 2 helper RNAs expressing the structuralproteins of the VEE virus. The cell culture supernatants were harvestedapproximately 30 hours after transfection and the replicon particleswere concentrated and purified by centrifugation through a 20% sucrosecushion. The pellets containing the packaged replicon particles weresuspended in PBS and the titers were determined by infecting Vero cellswith serial dilutions of the replicon particles and enumerating theinfected cells by indirect immunofluorescence with antibodies specificfor the Ebola proteins.

Immunoprecipitation of Ebola Virus Proteins Expressed from VEE VirusReplicons

BHK cells were transfected with either the Ebola virus GP, NP, VP24,VP30, VP35, or VP40 replicon RNAs. At 24 h post-transfection, theculture medium was replaced with minimal medium lacking cysteine andmethionine, and proteins were labeled for 1 h with ³⁵S-labeledmethionine and cysteine. Cell lysates or supernatants (supe) werecollected and immunoprecipitated with polyclonal rabbit anti-Ebola virusserum bound to protein A beads. ³⁵S-labeled Ebola virus structuralproteins from virions grown in Vero E6 cells were alsoimmunoprecipitated as a control for each of the virion proteins.Immunoprecipitated proteins were resolved by electrophoresis on an 11%SDS-polyacrylamide gel and were visualized by autoradiography.

Vaccination of Mice with VEE Virus Replicons

Groups of 10 BALB/c or C57BL/6 mice per experiment were subcutaneouslyinjected at the base of the neck with 2×10⁶ focus-forming units of VRPsencoding the Ebola virus genes. As controls, mice were also injectedwith either a control VRP encoding the Lassa nucleoprotein (NP) or withPBS. For booster inoculations, animals received identical injections at1 month intervals. Data are recorded as the combined results of 2 or 3separate experiments.

Ebola Infection of Mice

One month after the final booster inoculation, mice were transferred toa BSL-4 containment area and challenged by intraperitoneal (ip)inoculation of 10 plaque-forming units (pfu) of mouse-adapted Ebolavirus (approximately 300 times the dose lethal for 50% of adult mice).The mice were observed daily, and morbidity and mortality were recorded.Animals surviving at day 21 post-infection were injected again with thesame dose of virus and observed for another 21 days.

In some experiments, 4 or 5 mice from vaccinated and control groups wereanesthetized and exsanguinated on day 4 (BALB/c mice) or day 5 (C57BL/6mice) following the initial viral challenge. The viral titers inindividual sera were determined by plaque assay.

Passive Transfer of Immune Sera to Naive Mice.

Donor sera were obtained 28 days after the third inoculation with 2×10⁶focus-forming units of VRPs encoding the indicated Ebola virus gene, thecontrol Lassa NP gene, or from unvaccinated control mice. One mL ofpooled donor sera was administered intraperitoneally (ip) to naive,syngeneic mice 24 h prior to intraperitoneal challenge with 10 pfu ofmouse-adapted Ebola virus.

Vaccination and Challenge of Guinea Pigs.

EboGPVRP or EboNPVRP (1×10⁷ focus-forming units in 0.5 ml PBS) wereadministered subcutaneously to inbred strain 2 or strain 13 guinea pigs(300-400 g). Groups of five guinea pigs were inoculated on days 0 and 28at one (strain 2) or two (strain 13) dorsal sites. Strain 13 guinea pigswere also boosted on day 126. One group of Strain 13 guinea pigs wasvaccinated with both the GP and NP constructs. Blood samples wereobtained after vaccination and after viral challenge. Guinea pigs werechallenged on day 56 (strain 2) or day 160 (strain 13) by subcutaneousadministration of 1000 LD₅₀ (1×10⁴ PFU) of guinea pig-adapted Ebolavirus. Animals were observed daily for 60 days, and morbidity(determined as changes in behavior, appearance, and weight) and survivalwere recorded. Blood samples were taken on the days indicated afterchallenge and viremia levels were determined by plaque assay.

Virus Titration and Neutralization Assay.

Viral stocks were serially diluted in growth medium, adsorbed ontoconfluent Vero E6 cells in 6- or 12-well dishes, incubated for 1 hour at37° C., and covered with an agarose overlay (Moe, J. et al. (1981) J.Clin. Microbiol. 13:791-793). A second overlay containing 5% neutral redsolution in PBS or agarose was added 6 days later, and plaques werecounted the following day. Pooled pre-challenge serum samples from someof the immunized groups were tested for the presence ofEbola-neutralizing antibodies by plaque reduction neutralization assay.Aliquots of Ebola virus in growth medium were mixed with serialdilutions of test serum, or with normal serum, or medium only, incubatedat 37° C. for 1 h, and used to infect Vero E6 cells. Plaques werecounted 1 week later.

Cytotoxic T Cell Assays.

BALB/c and C57BL/6 mice were inoculated with VRPs encoding Ebola virusNP or VP24 or the control Lassa NP protein. Mice were euthanized atvarious times after the last inoculation and their spleens removed. Thespleens were gently ruptured to generate single cell suspensions. Spleencells (1×10⁶/ml) were cultured in vitro for 2 days in the presence of10-25 □M of peptides synthesized from Ebola virus NP or VP24 amino acidsequences, and then for an additional 5 days in the presence of peptideand 10% supernatant from concanavalin A-stimulated syngeneic spleencells. Synthetic peptides were made from Ebola virus amino acidsequences predicted by a computer algorithm (HLA Peptide BindingPredictions, Parker, K. C., et al. (1994) J. Immunol. 152:163) to have alikelihood of meeting the MHC class I binding requirements of the BALB/c(H-2^(d)) and C57BL/6 (H-2^(b)) haplotypes. Only 2 of 8 peptidespredicted by the algorithm and tested to date have been identified ascontaining CTL epitopes. After in vitro restimulation, the spleen cellswere tested in a standard ⁵¹chromium-release assay well known in the art(see, for example, Hart et al. (1991) Proc. Natl. Acad. Sci. USA 88:9449-9452). Percent specific lysis of peptide-coated, MHC-matched ormismatched target cells was calculated as:

$\frac{{{Experimental}\mspace{14mu}{cpm}} - {{Spontaneous}\mspace{14mu}{cpm} \times 100}}{{{Maximum}\mspace{14mu}{cpm}} - {{Spontaneous}\mspace{14mu}{cpm}}}$

Spontaneous cpm are the number of counts released from target cellsincubated in medium. Maximum cpm are obtained by lysing target cellswith 1% Triton X-100. Experimental cpm are the counts from wells inwhich target cells are incubated with varying numbers of effector (CTL)cells. Target cells tested were L5178Y lymphoma or P815 mastocytomacells (MHC matched to the H2^(d) BALB/c mice and EL4 lymphoma cells (MHCmatched to the H2^(b) C57BL/6 mice). The effector:target (E:T) ratiostested were 25:1, 12:1, 6:1 and 3:1.

EXAMPLE 1

Survival of Mice Inoculated with VRPs Encoding Ebola Proteins

Mice were inoculated two or three times at 1 month intervals with 2×10⁶focus-forming units of VRPs encoding individual Ebola virus genes, orLassa virus NP as a control, or with phosphate buffered saline (PBS).Mice were challenged with 10 pfu of mouse-adapted Ebola virus one monthafter the final immunization. The mice were observed daily, andmorbidity and mortality data are shown in Table 1A for BALB/c mice andTable 1B for C57BL/6 mice. The viral titers in individual sera of somemice on day 4 (BALB/c mice) or day 5 (C57BL/6 mice) following theinitial viral challenge were determined by plaque assay.

TABLE 1 Survival Of Mice Inoculated With VRPs Encoding Ebola ProteinsVRP #Injections S/T¹ (%) MDD² V/T³ Viremia⁴ A. BALB/c Mice EboNP 3 30/30(100%) 5/5 5.2 2 19/20 (95%) 7 5/5 4.6 EboGP 3 15/29 (52%) 8 1/5 6.6 214/20 (70%) 7 3/5 3.1 EboVP24 3 27/30 (90%) 8 5/5 5.2 2 19/20 (95%) 64/4 4.8 EboVP30 3 17/20 (85%) 7 5/5 6.2 2 11/20 (55%) 7 5/5 6.5 EboVP353 5/19 (26%) 7 5/5 6.9 2 4/20 (20%) 7 5/5 6.5 EboVP40 3 14/20 (70%) 85/5 4.6 2 17/20 (85%) 7 5/5 5.6 LassaNP 3 0/29 (0%) 7 5/5 8.0 2 0/20(0%) 7 5/5 8.4 none(PBS) 3 1/30 (3%) 6 5/5 8.3 2 0/20 (0%) 6 5/5 8.7 B.C57BL/6 Mice EboNP 3 15/20 (75%) 8 5/5 4.1 2 8/10 (80%) 9 ND⁵ ND EboGP 319/20 (95%) 10 0/5 — 2 10/10 (100%) — ND ND EboVP24 3 0/20 (0%) 7 5/58.6 EboVP30 3 2/20 (10%) 8 5/5 7.7 EboVP35 3 14/20 (70%) 8 5/5 4.5EboVP40 3 1/20 (5%) 7 4/4 7.8 LassaNP 3 1/20 (5%) 7 4/4 8.6 2 0/10 (0%)7 ND ND none(PBS) 3 3/20 (15%) 7 5/5 8.6 2 0/10 (0%) 7 ND ND ¹S/T,Survivors/total challenged. ²MDD, Mean day to death ³V/T, Number of micewith viremia/total number tested. ⁴Geometric mean of Log₁₀ viremiatiters in PFU/mL. Standard errors for all groups were 1.5 or less,except for the group of BALB/c mice given 2 inoculations of EboGP, whichwas 2.2. ⁵ND, not determined.

EXAMPLE 2

VP24-Immunized BALB/c Mice Survive a High-Dose Challenge with EbolaVirus

BALB/c mice were inoculated two times with 2×10⁶ focus-forming units ofEboVP24VRP. Mice were challenged with either 1×10³ pfu or 1×10⁵ pfu ofmouse-adapted Ebola virus 1 month after the second inoculation.Morbidity and mortality data for these mice are shown in Table 2.

TABLE 2 VP24-Immunized BALB/c Mice Survive A High-Dose Challenge WithEbola virus Replicon Challenge Dose Survivors/Total EboVP24 1 × 10³ pfu5/5 (3 × 10⁴ LD₅₀) EboVP24 1 × 10⁵ pfu 5/5 (3 × 10⁶ LD₅₀) None 1 × 10³pfu 0/4 (3 × 10⁴ LD₅₀) None 1 × 10⁵ pfu 0/3 (3 × 10⁶ LD₅₀)

EXAMPLE 3

Passive Transfer of Immune Sera Can Protect Naive Mice from a LethalChallenge of Ebola Virus

Donor sera were obtained 28 days after the third inoculation with 2×10⁶focus-forming units of VRPs encoding the indicated Ebola virus gene, thecontrol Lassa NP gene, or from unvaccinated control mice. One mL ofpooled donor sera was administered intraperitoneally (ip) to naive,syngeneic mice 24 h prior to intraperitoneal challenge with 10 pfu ofmouse-adapted Ebola virus.

TABLE 3 Passive Transfer of Immune Sera Can Protect Unvaccinated Micefrom a Lethal Challenge of Ebola Virus Specificity of Survivors/ MeanDay Donor Sera Total of Death A. BALB/c Mice Ebola GP 15/20  8 Ebola NP1/20 7 Ebola VP24 0/20 6 Ebola VP30 0/20 7 Ebola VP35 ND¹ ND Ebola VP400/20 6 Lassa NP 0/20 7 Normal mouse sera 0/20 6 B. C57BL/6 Mice Ebola GP17/20  7 Ebola NP 0/20 7 Ebola VP24 ND ND Ebola VP30 ND ND Ebola VP350/20 7 Ebola VP40 ND ND Lassa NP 0/20 7 Normal mouse sera O/20 7 ¹ND,not determined

EXAMPLE 4

Immunogenicity and Efficacy of VRepEboGP and VRepEboNP in Guinea Pigs.

EboGPVRP or EboNPVRP (1×10⁷ IU in 0.5 ml PBS) were administeredsubcutaneously to inbred strain 2 or strain 13 guinea pigs (300-400 g).Groups of five guinea pigs were inoculated on days 0 and 28 at one(strain 2) or two (strain 13) dorsal sites. Strain 13 guinea pigs werealso boosted on day 126. One group of Strain 13 guinea pigs wasvaccinated with both the GP and NP constructs. Blood samples wereobtained after vaccination and after viral challenge.

Sera from vaccinated animals were assayed for antibodies to Ebola byplaque-reduction neutralization, and ELISA. Vaccination with VRepEboGPor NP induced high titers of antibodies to the Ebola proteins (Table 4)in both guinea pig strains. Neutralizing antibody responses were onlydetected in animals vaccinated with the GP construct (Table 4).

Guinea pigs were challenged on day 56 (strain 2) or day 160 (strain 13)by subcutaneous administration of 1000 LD₅₀ (10⁴ PFU) of guineapig-adapted Ebola virus. Animals were observed daily for 60 days, andmorbidity (determined as changes in behavior, appearance, and weight)and survival were recorded. Blood samples were taken on the daysindicated after challenge and viremia levels were determined by plaqueassay. Strain 13 guinea pigs vaccinated with the GP construct, alone orin combination with NP, survived lethal Ebola challenge (Table 4).Likewise, vaccination of strain 2 inbred guinea pigs with the GPconstruct protected 3/5 animals against death from lethal Ebolachallenge, and significantly prolonged the mean day of death (MDD) inone of the two animals that died (Table 4). Vaccination with NP alonedid not protect either guinea pig strain.

TABLE 4 Immunogenicity and efficacy of VRepEboGP and VRepEboNP in guineapigs Survivors/ Viremia^(c) VRP ELISA^(a) PRNT₅₀ total(MDD^(b)) d7 d14A. Strain 2 guinea pigs GP 4.1 30 3/5 (13 + 2.8) 2.3 1.8 NP 3.9 <10 0/5(9.2 + 1.1) 3.0 — Mock <1.5 <10 0/5 (8.8 + 0.5) 3.9 — B. Strain 13guinea pigs GP 4.0 140 5/5 <2.0 <2.0 GP/NP 3.8 70 5/5 <2.0 <2.0 NP 2.8<10 1/5 (8.3 + 2.2) 4.6 — Lassa NP <1.5 <10 2/5 (8.3 + 0.6) 4.8 —^(a)Data are expressed as geometric mean titers, log₁₀. ^(b)MDD, meanday to death ^(c)Geometric mean of log₁₀ viremia titers in PFU/mL.Standard errors for all groups were 0.9 or less.

EXAMPLE 5

Induction of Murine CTL Responses to Ebola Virus NP and Ebola Virus VP24Proteins

BALB/c and C57BL/6 mice were inoculated with VRPs encoding Ebola virusNP or VP24. Mice were euthanized at various times after the lastinoculation and their spleens removed. Spleen cells (1×10⁶/ml) werecultured in vitro for 2 days in the presence of 10 to 25 □M of peptides,and then for an additional 5 days in the presence of peptide and 10%supernatant from concanavalin A-stimulated syngeneic spleen cells. Afterin vitro restimulation, the spleen cells were tested in a standard⁵¹chromium-release assay. Percent specific lysis of peptide-coated,MHC-matched or mismatched target cells was calculated as:

$\frac{{{Experimental}\mspace{14mu}{cpm}} - {{Spontaneous}\mspace{14mu}{cpm} \times 100}}{{{Maximum}\mspace{14mu}{cpm}} - {{Spontaneous}\mspace{14mu}{cpm}}}$In the experiments shown, spontaneous release did not exceed 15%.

TABLE 5 Induction of murine CTL responses to Ebola virus NP and Ebolavirus VP24 proteins. % Specific Lysis E:T ratio Mice, VRP¹ Peptide²Cell³ 25 BALB/c, VP24 None P815 55 BALB/c, VP24 SEQ ID NO: 25 P815 93C57BL/6, EboNP None EL4 2 C57BL/6, EboNP⁴ SEQ ID NO: 24 EL4 70 C57BL/6,EboNP LassaNP EL4 2 C57BL/6, LassaNP None L5178Y 1 C57BL/6, LassaNP SEQID NO: 24 L5178Y 0 C57BL/6, LassaNP None EL4 2 C57BL/6, LassaNP SEQ IDNO: 24 EL4 6 ¹Indicates the mouse strain used and the VRP used as the invivo immunogen. In vitro restimulation was performed using SEQ ID NO: 24peptide for BALB/c mice and SEQ ID NO: 23 for all C57BL/6 mice shown.²Indicates the peptide used to coat the target cells for the chromiumrelease assay. ³Target cells are MHC-matched to the effector cells,except for the L5178Y cells that are C57BL/6 mismatched. ⁴High levels ofspecific lysis (>40%) were also observed using E:T ratios of 12, 6, 3,or 1:1.

EXAMPLE 6

Induction of Murine T Cell Responses that Protect Against EbolaChallenge

Mice and injections. BALB/c and C57Bl/6 mice were injected sc with 2×10⁶IU of VEE virus replicons encoding either the individual Ebola genes orLassa NP (3 injections 1 month apart). The genes used to make repliconsare from the human Zaire76 virus. One month after the finalimmunization, mice were transferred to BSL-4 containment and challengedby ip inoculation of 10 or 1000 pfu (300 or 30000 LD₅₀) of mouse-adaptedEbola Zaire. This virus has amino acid changes in NP at nt 683 (S to G),VP35 at nt 3163 (A to V), VP24 at nt 10493 (T to I), and in L at nt14380 (F to L) and nt 16174 (I to V). There are three other nt changes,including an insertion in the intergenic region at nt 10343. GenBankaccession number AF499101.

T cell assays. Single cell suspensions were prepared from spleens bypassage through cell 70 μM strainers. Spleen cells were depleted oferythrocytes by treatment with buffered ammonium chloride solution andenumerated by trypan blue exclusion on a hemacytometer. For in vitrorestimulations, 1-5 μg peptide(s) and human recombinant IL-2 (10 U/ml,National Cancer Institute) were added to a cell density of 1×10⁶/ml andthe cultures incubated 4-7 days. For intracellular IFN-γ staining,splenocytes were cultured at 37° C. for 5 hr with 1-5 μg of peptide(s)or PMA (25 ng/ml) and ionomycin (1.25 ug/ml) in 100 μl complete mediumcontaining 10 μg/ml brefeldin A (BFA). After culture, the cells wereblocked with mAbs to FcRIII/II receptor and stained with αCD44 FITC andanti-CD8 Cychrome (Pharmingen, San Diego, Calif.) in PBS/BFA. The cellswere then fixed in 1% formaldehyde (Ted Pella, Redding, Calif.),permeabilized with PBS containing 0.5% saponin, and stained with αIFN-γPE (Pharmingen, San Diego, Calif.). The data were acquired using aFACSCalibur flow cytometer and analyzed with CELLQuest software(Becton-Dickinson. Cytotoxicity assays were performed using target cells(EL4, L5178Y) labeled with ⁵¹Cr (Na₂CrO₄; New England Nuclear, Boston,Mass.) and pulsed with peptide for 1.5 hours. Unpulsed target cells wereused as negative controls. Various numbers of effector cells wereincubated with 2500 target cells for 4 hours. Percentage specificrelease was calculated as: % specificrelease=(experimental−spontaneous)/(maximum−spontaneous)×100.Spontaneous release values were obtained by incubation of target cellsin medium alone and were routinely <10% of maximum release. Maximumrelease values were obtained by the addition of 100 μl 1% TritonX-100.

Adoptive transfer experiments. After in vitro restimulation, cells areFicoll purified, washed three times with 0.3M methyl-a-D-mannopyranosideand twice with complete media. Cells are counted and adjusted to25.0×10^6 cells/ml in endotoxin-free PBS. A total volume of 0.2 mls isgiven to each mouse by i.p. injection 4 hr before challenge with 1000PFU of mouse-adapted Ebola virus. Animals are observed and sickness ordeath is noted on daily charts.

As shown in Table A, this data identifies the protective mechanisminduced by VRP vaccination, showing the role of T cells. It indicatesthe ability to predict protection from in vitro assays, specifically theintracellular cytokine and chromium release assays. Thus, a positive ICCresult is reasonably predictive of conferred protection, even if theprotection is listed as incomplete; the rest of the data stronglyindicate protection. Notably, where the CTL sequences are conservedbetween Zaire and the other Ebola viruses, cross-protection mayreasonably be inferred. (The protection in Table A refers to adoptivetransfer of CTLs to unvaccinated mice before challenge, not thevaccination with a certain protein.)

EXAMPLE 7

Determination of Interference in Protection by Multiple Replicons

The purpose of this experiment was to determine if multiple VEEreplicons that do not provide complete protection (VP24, 30, 40 in BL/6mice) will interfere with a protective replicon Ebola NP that hasdefined CTL epitopes. Co-administration did not interfere with theinduction of protection.

Vaccine Survival VRep EBOV NP, VP24, VP30, VP40 10/10 VRep EBOV NP 6/6PBS 0/7

The CTLs to NP are CD8(+),and recognize epitopes SEQ ID NO:24, 26 and27. Evaluation of the ability to lyse peptide-pulsed target cells wasassessed using spleen cells from mice vaccinated with the EBOC VRepNPalone, or all four replicons (NP, VP24, VP30 and VP40). Althoughresponses were somewhat lower in the mice receiving four replicons, thethreshold of immunity was maintained.

% Lysis of target cells coated with peptide (background on untreatedtarget cells is subtracted) Vaccine Effector/target ratio NP-1 NP-8NP-17 NP 100:1  55 31 64 50:1 61 23 45 25:1 67 15 31 12:1 51 13 21 Mix100:1  41 40 45 50:1 37 32 28 25:1 26 33 19 12:1 19 18 12

EXAMPLE 8

Improved Efficacy Induced by a Cocktail Formulation of Suboptimal EBOVVrep

In studies where we examined the protective efficacy of repliconsindividually, we observed that some replicons (such as VP30, VP24 andVP40) protected fewer than 100% of the mice. When protection was lessthan 50%, we suggested that the protein was not particularly protectivefor that mouse strain. However, in some cases, we did observe that20-30% of the mice survived, suggesting that we might be able tooptimize our vaccine strategy to provide protection with those proteins.As we are evaluating a cocktail formulation, we approached this issue byinjecting mice with combinations of the three VP replicons than had poorefficacy in C57Bl/6 mice.

C57BL/6 mice were injected SC at the base of the neck with 2.0×10⁶packaged VEE virus replicon particles for each Ebola VP protein, thenrested for 27 days and then boosted twice at days 28 and 56. On day 84,mice were injected intraperitoneally with approximately 3×10⁴ LD50 (1000PFU) of mouse-adapted Ebola virus.

Strain Vaccine Survival C57BL/6 VRep VP35 30/30 C57BL/6 VReps VP35,VP30, VP24, VP40 30/30 C57BL/6 Medium  0/23 C57BL/6 VReps VP30, VP24,VP40 28/30

The data indicate that combining the three VP replicons providedsignificantly better protection than when we administered them singly.Of note, the VP24 replicon has never protected a single C57BL/6 mousewhen administered alone and is not likely contributing to protection.However, importantly, its inclusion in the formulation also does notinterfere with induction of protective responses to the other VPs.

These data shows that a cocktail formulation may be a preferred vaccinebecause it induces a broader array of T cells (i.e. CTLs to multipleproteins) and that, together, these may meet the threshold needed forprotection. We expect the same phenomenon will apply to non-humanprimate studies. This also provides support for the inclusion ofmultiple Ebola peptides/proteins in a cocktail formulation. As anexample, if a human infected with Ebola needs 1 million effectors, butvaccination induces only 400,000 to each protein, it may be additive tohave 1.2 million spread across 3 proteins. Otherwise, waiting one dayfor that person's cells to divide to 800,000 and a second day to cross 1million—but that would likely be too late for survival.

Ebola Zaire 1976 (Mayinga) virus causes acute hemorrhagic fevercharacterized by high mortality. There are no current vaccines oreffective therapeutic measures to protect individuals who are exposed tothis virus. In addition, it is not known which genes are essential forevoking protective immunity and should therefore be included in avaccine designed for human use. In this study, the GP, NP, VP24, VP30,VP35, and VP40 virion protein genes of the Ebola Zaire 1976 (Mayinga)virus were cloned and inserted into a Venezuelan equine encephalitis(VEE) virus replicon vector (VRep) as shown in FIGS. 2A and 2B. TheseVReps were packaged as VEE replicon particles (VRPs) using the VEE virusstructural proteins provided as helper RNAs, as shown in FIG. 3. Thisenables expression of the Ebola virus proteins in host cells. The Ebolavirus proteins produced from these constructs were characterized invitro and were shown to react with polyclonal rabbit anti-Ebola virusantibodies bound to Protein A beads following SDS gel electrophoresis ofimmunoprecipitated proteins (FIG. 4).

The Ebola virus genes were sequenced from the VEE replicon clones andare listed here as SEQ ID NO:1 (GP), 2 (NP), 3 (VP24), 4 (VP30), 5(VP35), 6 (VP40), and 7 (VP30#2) as described below. The correspondingamino acid sequences of the Ebola proteins expressed from thesereplicons are listed as SEQ ID NO: 17, 18, 19, 20, 21, 22, and 23,respectively. Changes in the DNA sequence relative to the sequencepublished by Sanchez et al. (1993) are described relative to thenucleotide (nt) sequence number from GenBank (accession number L11365).

The sequence we obtained for Ebola virus GP (SEQ ID NO:1) differed fromthe GenBank sequence by a transition from A to G at nt 8023. Thisresulted in a change in the amino acid sequence from Ile to Val atposition 662 (SEQ ID NO: 17).

The DNA sequence we obtained for Ebola virus NP (SEQ ID NO:2) differedfrom the GenBank sequence at the following 4 positions: insertion of a Cresidue between nt 973 and 974, deletion of a G residue at nt 979,transition from C to T at nt 1307, and a transversion from A to C at nt2745. These changes resulted in a change in the protein sequence fromArg to Glu at position 170 and a change from Leu to Phe at position 280(SEQ ID NO: 18).

The Ebola virus VP24 (SEQ ID NO:3) gene differed from the GenBanksequence at 6 positions, resulting in 3 nonconservative changes in theamino acid sequence. The changes in the DNA sequence of VP24 consistedof a transversion from G to C at nt 10795, a transversion from C to G atnt 10796, a transversion from T to A at nt 10846, a transversion from Ato T at nt 10847, a transversion from C to G at nt 11040, and atransversion from C to G at nt 11041. The changes in the amino acidsequence of VP24 consisted of a Cys to Ser change at position 151, a Leuto His change at position 168, and a Pro to Gly change at position 233(SEQ ID NO: 19).

We have included 2 different sequences for the Ebola virus VP30 gene(SEQ ID NOS:4 and SEQ ID NO:7). Both of these sequences differ from theGenBank sequence by the insertion of an A residue in the upstreamnoncoding sequence between nt 8469 and 8470 and an insertion of a Tresidue between nt 9275 and 9276 that results in a change in the openreading frame of VP30 and VP30#2 after position 255 (SEQ ID NOS:20 andSEQ ID NO:23). As a result, the C-terminus of the VP30 protein differssignificantly from that previously reported. In addition to these 2changes, the VP30#2 gene in SEQ ID NO:23 contains a conservativetransition from T to C at nt 9217. Because the primers originally usedto clone the VP30 gene into the replicon were designed based on theGenBank sequence, the first clone that we constructed (SEQ ID NO:4) didnot contain what we believe to be the authentic C-terminus of theprotein. Therefore, in the absence of the VP30 stop codon, theC-terminal codon was replaced with 37 amino acids derived from thevector sequence. The resulting VP30 construct therefore differed fromthe GenBank sequence in that it contained 32 amino acids of VP30sequence (positions 256 to 287, SEQ ID NO:20) and 37 amino acids ofirrelevant sequence (positions 288 to 324, SEQ ID NO:20) in the place ofthe C-terminal 5 amino acids reported in GenBank. However, inclusion of37 amino acids of vector sequence in place of the C-terminal amino acid(Pro, SEQ ID NO:23) did not inhibit the ability of the protein to serveas a protective antigen in BALB/c mice. We have also determined that aVEE replicon construct (SEQ ID NO:7), which contains the authenticC-terminus of VP30 (VP30#2, SEQ ID NO:23), is able to protect miceagainst a lethal Ebola challenge.

The DNA sequence for Ebola virus VP35 (SEQ ID NO:5) differed from theGenBank sequence by a transition from T to C at nt 4006, a transitionfrom T to C at nt 4025, and an insertion of a T residue between nt 4102and 4103. These sequence changes resulted in a change from a Ser to aPro at position 293 and a change from Phe to Ser at position 299 (SEQ IDNO:21). The insertion of the T residue resulted in a change in the openreading frame of VP35 from that previously reported by Sanchez et al.(1993) following amino acid number 324. As a result, Ebola virus VP35encodes for a protein of 340 amino acids, where amino acids 325 to 340(SEQ ID NO:21) differ from and replace the C-terminal 27 amino acids ofthe previously published sequence.

Sequencing of VP30 and VP35 was also performed on RT/PCR products fromRNA derived from cells that were infected with Ebola virus 1976, Ebolavirus 1995 or the mouse-adapted Ebola virus. The changes noted above forthe VRep constructs were also found in these Ebola viruses. Thus, webelieve that these changes are real events and not artifacts of cloning.

The Ebola virus VP40 differed from the GenBank sequence by atransversion from a C to G at nt 4451 and a transition from a G to A atnt 5081. These sequence changes did not alter the protein sequence ofVP40 (SEQ ID NO:22) from that of the published sequence.

To evaluate the protective efficacy of individual Ebola virus proteinsand to determine whether the major histocompatibility (MHC) genesinfluence the immune response to Ebola virus antigens, twoMHC-incompatible strains of mice were vaccinated with VRPs expressing anEbola protein. As controls for these experiments, some mice wereinjected with VRPs expressing the nucleoprotein of Lassa virus or wereinjected with phosphate-buffered saline (PBS). Following Ebola viruschallenge, the mice were monitored for morbidity and mortality, and theresults are shown in Table 1.

The GP, NP, VP24, VP30, and VP40 proteins of Ebola virus generatedeither full or partial protection in BALB/c mice, and may therefore beuseful components of a vaccine for humans or other mammals. Vaccinationwith VRPs encoding the NP protein afforded the best protection. In thiscase, 100% of the mice were protected after three inoculations and 95%of the mice were protected after two inoculations. The VRP encoding VP24also protected 90% to 95% of BALB/c mice against Ebola virus challenge.In separate experiments (Table 2), two or three inoculations with VRPsencoding the VP24 protein protected BALB/c mice from a high dose (1×10⁵plaque—forming units (3×10⁶ LD50)) of mouse-adapted Ebola virus.

Example 1 shows that vaccination with VRPs encoding GP protected 52-70%of BALB/c mice. The lack of protection was not due to a failure torespond to the VRP encoding GP, as all mice had detectable Ebolavirus-specific serum antibodies after vaccination. Improved results werelater seen, which are thought to be dose-dependent. Further, as shown inExamples 6-8, combining suboptimal formulations gives dramaticallybetter protection.

Also in Example 1, some protective efficacy was further observed inBALB/c mice vaccinated two or three times with VRPs expressing the VP30protein (55% and 85%, respectively),or the VP40 protein (70% and 80%,respectively). The VP35 protein was not efficacious in the BALB/c mousemodel, as only 20% and 26% of the mice were protected after either twoor three doses, respectively. Again, improved results were later seen,which are thought to be dose-dependent; and we found that combiningsuboptimal formulations gives dramatically better protection (e.g.,combination of VP24, VP30 and VP40).

Geometric mean titers of viremia were markedly reduced in BALB/c micevaccinated with VRPs encoding Ebola virus proteins after challenge withEbola virus, indicating an ability of the induced immune responses toreduce virus replication (Table 1A). In this study, immune responses tothe GP protein were able to clear the virus to undetectable levelswithin 4 days after challenge in some mice.

When the same replicons were examined for their ability to protectC57BL/6 mice from a lethal challenge of Ebola virus, only the GP, NP,and VP35 proteins were efficacious (Table 1B). The best protection, 95%to 100%, was observed in C57BL/6 mice inoculated with VRPs encoding theGP protein. Vaccination with VRPs expressing NP protected 75% to 80% ofthe mice from lethal disease. In contrast to what was observed in theBALB/c mice, the VP35 protein was the only VP protein able tosignificantly protect the C57BL/6 mice. In this case, 3 inoculationswith VRPs encoding VP35 protected 70% of the mice from Ebola viruschallenge. The reason behind the differences in protection in the twomouse strains is believed to be due to the ability of the immunogens tosufficiently stimulate the cellular immune system. As with the BALB/cmice, the effects of the induced immune responses were also observed inreduced viremias and, occasionally, in a prolonged time to death ofC57BL/6 mice.

Example 4 shows that VRPs expressing Ebola virus GP or NP were alsoevaluated for protective efficacy in a guinea pig model. Sera fromvaccinated animals were assayed for antibodies to Ebola by westernblotting, IFA, plaque-reduction neutralization, and ELISA. Vaccinationwith either VRP (GP or NP) induced high titers of antibodies to theEbola proteins (Table 4) in both guinea pig strains. We later found thatVP40 induced high titers (4 logs) in mice. Neutralizing antibodyresponses were only detected in animals vaccinated with the VRPexpressing GP (Table 4).

As shown in Example 4, vaccination of strain 2 inbred guinea pigs withthe GP construct protected 3/5 animals against death from lethal Ebolachallenge, and significantly prolonged the mean day of death in one ofthe two animals that died (Table 4). All of the strain 13 guinea pigsvaccinated with the GP construct, alone or in combination with NP,survived lethal Ebola challenge (Table 4). Vaccination with NP alone didnot protect either guinea pig strain from challenge with the guineapig-adapted Ebola virus. Of note, guinea pigs are also inbred, and thefailure of NP to protect may indicate that they could not respond withappropriate T cells, but could make protective antibodies to GP. This isfurther support for our preferred embodiments including multiplepeptides and proteins, and even all six of the Ebola proteins.

As shown in Example 3, to identify the immune mechanisms that mediateprotection against Ebola virus and to determine whether antibodies aresufficient to protect against lethal disease, passive transfer studieswere performed. One mL of immune sera, obtained from mice previouslyvaccinated with one of the Ebola virus VRPs, was passively administeredto unvaccinated mice 24 hours before challenge with a lethal dose ofmouse-adapted Ebola virus. Antibodies to GP, but not to NP or the VPproteins, protected mice from an Ebola virus challenge (Table 3).Antibodies to GP protected 75% of the BALB/c mice and 85% of the C57BL/6mice from death. When the donor sera were examined for their ability toneutralize Ebola virus in a plaque-reduction neutralization assay, a1:20 to 1:40 dilution of the GP-specific antisera reduced the number ofviral plaque-forming units by at least 50% (data not shown). Incontrast, antisera to the NP and VP proteins did not neutralize Ebolavirus at a 1:20 or 1:40 dilution. These results are consistent with thefinding that GP is the only viral protein found on the surface of Ebolavirus, and is likely to induce virus-neutralizing antibodies.

As shown in Examples 5 and 6, cince the NP and VP proteins of Ebolavirus are internal virion proteins to which antibodies are notsufficient for protection, it is likely that cytotoxic T lymphocytes(CTLs) are also important for protection against Ebola virus. Theinventors investigated cellular immune responses to individual Ebolavirus proteins expressed from VRPs identified CTL responses to the VP24and NP proteins (Table 5). One CTL epitope that we identified for theEbola virus NP is recognized by C57BL/6 (H-2^(b)) mice, and has an aminoacid sequence of, or contained within, the following 11 amino acids:VYQVNNLEEIC (SEQ ID NO:24). Vaccination with EboNPVRP and in vitrorestimulation of spleen cells with this peptide consistently inducesstrong CTL responses in C57BL/6 (H-2^(b)) mice. In vivo vaccination toEbola virus NP is required to detect the CTL activity, as evidenced bythe failure of cells from C57BL/6 mice vaccinated with Lassa NP todevelop lytic activity to peptide (SEQ ID NO:24) after in vitrorestimulation with it. Specific lysis has been observed using very loweffector:target ratios (<2:1). This CTL epitope is H-2^(b) restricted inthat it is not recognized by BALB/c (H-2^(d)) cells treated the same way(data not shown), and H-2^(b) effector cells will not lyseMHC-mismatched target cells coated with this peptide.

A CTL epitope in the VP24 protein was also identified. It is recognizedby BALB/c (H-2^(d)) mice, and has an amino acid sequence of, orcontained within, the following 23 amino acids: LKFINKLDALLVVNYNGLLSSIF(SEQ ID NO:25). In the data shown in Table 5, high (>90%) specific lysisof P815 target cells coated with this peptide was observed. Thebackground lysis of cells that were not peptide-coated was also high(>50%), which is probably due to the activity of natural killer cells.We are planning to repeat this experiment using the L5178Y target cells,which are not susceptible to natural killer cells. This shows that CTLsmediated protection, which is further demonstrated by the evidence inExamples 6, 7 and 8.

1. An isolated glycoprotein (GP) Ebola peptide comprising the sequencespecified in SEQ ID NO:29, or consisting of an isolated peptide fragmentof SEQ ID NO:29 comprising at least 9 consecutive amino acids whichincludes the amino acid threonine corresponding to position 14 in SEQ IDNO:29.
 2. An isolated peptide fragment consisting of YFGPAAEGI (SEQ IDNO:42).
 3. A composition comprising the isolated peptide of SEQ ID NO:29 or an isolated peptide consisting of SEQ ID NO:42, in an effectiveimmunogenic amount in a pharmaceutically acceptable carrier and/oradjuvant.
 4. An immunogenic composition comprising the isolated peptideof SEQ ID NO: 29 or an isolated peptide consisting of SEQ ID NO:42, inan effective immunogenic amount in a pharmaceutically acceptable carrierand/or adjuvant.
 5. A composition comprising recombinant virus repliconparticles expressing the peptide of SEQ ID NO:29 or an isolated peptideconsisting of SEQ ID NO: 42, in an effective immunogenic amount in apharmaceutically acceptable carrier and/or adjuvant.
 6. The compositionof claim 5, wherein the recombinant virus replicon particles areproduced from a replicon vector selected from the group consisting ofVenezuelan Equine Encephalitis (VEE) virus, eastern equine encephalitis,western equine encephalitis, Semliki forest and Sindbis.